STTR_Developing Nanopieces 7. Project Summary/Abstract The bottleneck of developing new RNAi (RNA Interference) drugs is the lack of highly efficient and non-toxic RNA delivery to non-liver tissues in vivo. It is especially challenging to deliver negatively-charged nucleic acid into avascular, dense, and negatively-charged matrix in hard-to-reach tissues including joint cartilage. NanoDe Therapeutics, Inc. (NanoDe) is a development stage company dedicated to developing delivery of RNAi therapeutics, thereby creating more effective drugs. NanoDe is founded on a novel platform RNA delivery technology termed NanopiecesTM, a novel biomimetic nanomaterial derived from a small molecule JBAK, Janus-Base with Amine or lysine (K). Through self-interaction of its biocompatible Janus-Base units mimicking DNA base pairs, JBAK forms non-covalent nanotubes (NT) with positively charged amine or lysine on the surface. JBAK NT further assembles with siRNA to form JBAK NP, thereby encapsulating negatively charged siRNA into positively charged NP. Our preliminary data has shown that 1) NPs can penetrate matrix-rich tissues that conventional vehicles cannot, and release siRNA therapeutics intracelluarly in high efficiency to modify otherwise untreatable diseases; 2) NP delivered RNAi therapeutics has achieved successful outcomes in the treatment of multiple diseases including rheumatoid arthritis, a rare solid tumor called chondrosarcoma, and post-traumatic osteoarthritis (PTOA) in animal models respectively; and 3) NP has excellent biocompatibility and biodegradability, which are critical for maintaining minimal toxicity in vivo. The goal of this Phase I application is to further develop NP delivery technology by using PTOA as a ?use case? disease. The central hypothesis is that the optimal positive charge of NP is one of the critical parameters to enable its penetration into negatively charged cartilage matrix, endocytosis into chondrocytes, and release siRNA to inhibit disease gene expression, thereby achieving significant therapeutic effects on PTOA. This hypothesis will be tested with the two aims: 1) Determining the optimal charge of NP to enable its penetration and retention within cartilage tissue, transfection into chondrocytes, and inhibiting matrix proteinase ADAMTS-5 gene expression in chondrocytes, respectively; and 2) Determining the optimal charge of NP to enable its intra- articular delivery and long half-life within joint, and achieve significant therapeutic outcomes in a PTOA animal model. The proposed research is innovative because: 1) Essentially different from conventional delivery vehicles, Nanopiece is a non-covalent vehicle presenting unique advantages, such as versatility in dimensions and surface charge, affinity to extracellular matrix, excellent biodegradability and biocompatibility. 2) For the first time, we identify important technology parameters of NP required for successful siRNA delivery including the optimal vehicle surface charge for matrix penetration, cell transfection, and gene knockdown. 3) The technology breakthrough enlightens a therapeutic approach to deliver RNAi for treatment of multiple diseases including PTOA.